Receptor tyrosine kinases are involved in stimulating the growth of many cancers. In general, receptor tyrosine kinases are glycoproteins which consist of (1) an extracellular domain that is able to bind with a specific ligand, (2) a transmembrane region, (3) a juxtamembrane domain which may regulate the receptor activity by, for instance, protein phosphorylation, (4) a tyrosine kinase domain that is the enzymatic component of the receptor, and (5) a carboxyterminal tail. The ErbB family of type 1 receptor tyrosine kinases constitute one important class of receptors because of their importance in mediating cell growth, differentiation and survival in many solid tumors. Members of this receptor family include ErbB1 (also known as HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4). More than a dozen ligands interact with the ErbB-family receptors. For example, EGF, Transforming Growth Factor α (TGFα), and amphiregulin all bind to ErbB1. Isoforms of neuregulin, also known as Heregulin and Neu Differentiation Factor (NDF) have specific affinity for ErbB3 and ErbB4. Ligands such as betacellulin, heparin-binding EGF and epiregulin bind to both ErbB1 and ErbB4.
It is becoming clear that over expression of ErbB activating ligands can cause uncontrolled cellular proliferation similar to that of a deregulated receptor. In such cases, interference with the binding of the activating ligand to its receptor may provide an effective therapeutic strategy or that could accentuate current receptor based or other therapies. Therapeutics that interfere with ligand binding to ErbB3 may be particularly effective. ErbB3 differs from the other receptors in the EGFR family because its tyrosine kinase domain is functionally inactive; however, ErbB2/ErbB3 heterodimers transmit the most potent mitogenic signals of any homo- or heterodimer combination of the ErbB family. Therefore, ErbB3 is an important target, yet one that cannot be inhibited through small molecules that target the kinase region. Since ErbB3 requires an activating ligand, such as heregulin or NDF, before activated heterodimers can form, molecules that can interfere with the binding of ErbB3 receptor ligands could be used to block or interfere with the formation of ErbB dimers and heterodimers. One example of such a molecule would be a molecule that has ligand binding affinity and can therefore “trap” ligands and effectively reduce their concentration so that they cannot activate the ErbB3 receptor. In addition to ErbB3 ligands, the other known ErbB receptor ligands, have similar effects to varying degrees. Thus, binding molecules that can trap and sequester the full spectrum of ErbB ligands may be of even more use in the treatment of cancer.
Several therapeutics exist that have attempted this trapping or “decoy” strategy. For example, Enbrel™ (etanercept-Amgen) is a soluble, modified version of the TNFR receptor that binds and traps the pro-inflammatory ligand TNFα. In addition, a soluble fusion protein of the VEGFR1 and VEGFR2 receptors, called a VEGF Trap, is currently in clinical trials for the treatment of both macular degeneration and several forms of cancer (Regeneron Pharmaceuticals). An ErbB3 trap has also shown potency in vitro at enhancing the effects of a dual EGFR/ErbB2 inhibitor and reversed GW2974 (a small molecule inhibitor of ErbB1 and ErbB2) resistance in cells treated with NDF.
All currently approved ErbB inhibitors target either EGFR, ErbB2, ErbB3, ErbB4 or combinations of all 4 receptors. However, no therapeutic is known that interferes with the binding of ligands to multiple ErbB receptors simultaneously. Clearly, new binding molecules are needed that can be used to sequester receptor ligands, such as ErbB ligands, and thereby block ligand binding to multiple ErbB receptors and subsequent receptor activation. Binding molecules capable of binding all known ErbB ligands would be particularly useful.
A number of binding studies have been carried out to determine regions of ErbB3 that are important to the binding of its ligand, heregulin. Singer, et. al. (2001), J. Biol. Chem. 276, 44266-44274. Other studies using chimeric receptors have identified the relative contributions of the extracellular domains of ErbB1 and ErbB4 to ligand-specific signaling. Kim, et. al. (2002), Eur. J. Biochem. 269, 2323-2329. These studies reveal that neuregulin binding to ErbB4 depends much more on domain I than on domain III and that domain III of ErbB1 is primarily important for EGF binding. However, these studies were conducted on full length receptors which span the entire length of the receptors including the transmembrane and cytoplasmic domains. These large molecules present manufacturing and administration problems potentially leading to lower therapeutic efficacy.